Engineered recombinant human paraoxonase 1 (rHuPON1) purified from Escherichia coli protects against organophosphate poisoning

Abstract

The high-density lipoprotein-associated enzyme paraoxonase 1 (PON1) hydrolyzes lactones, aromatic esters, and neurotoxic organophosphorus (OP) compounds, including insecticide metabolites and nerve agents. Experiments with mice lacking PON1 (PON1(-/-) mice) have established that plasma PON1 protects against chlorpyrifos/chlorpyrifos-oxon and diazinon/diazoxon (DZO) exposure but does not protect against parathion/paraoxon or nerve agents. The catalytic efficiency of PON1 determines whether or not it will protect against a given OP exposure. Expression of active recombinant human PON1 (rHuPON1) in Escherichia coli provides a system in which PON1 can be engineered to achieve a catalytic efficiency sufficient to protect against or treat specific OP exposures. Here, we describe the generation of highly purified engineered rHuPON1(K192) that protects against DZO exposure when injected into PON1(-/-) mice. The injected rHuPON1 is nontoxic, persists in serum for at least 2 days after injection, and provides protection against DZO exposures of at least three times the median lethal dose value.

Fig. 1.

Protein (A₂₈₀) and activity monitoring during chromatographic purification of rHuPON1_K192. Absorbance measurements at 280 nm (gray lines) allowed for continuous monitoring of protein elution from chromatography media, whereas AREase activity (black data points and line) measurements of column fractions allowed monitoring of rHuPON1_K192. (A) First diethylaminoethyl (DEAE) column. (B) Second DEAE column. (C) hydroxyapatite (HA) column. (D) Third DEAE column. (E) Hydrophobic interaction chromatography (HIC) column. (F) Gel filtration column. (G) Fourth DEAE column.

Fig. 2.

SDS/PAGE gel analysis of the pooled column fractions from the purification of rHuPON1s. (A) rHuPON1_K192 variant. Lane 1, molecular weight markers (kDa); lane 2, starting extract; lane 3, DEAE I; lane 4, G-25 desalting column; lane 5, DEAE II; lane 6, HA; lane 7, DEAE III; lane 8, HIC; lane 9, gel filtration; lane 10, DEAE IV. (B) rHuPON1_R192 alloform. Lane 11, molecular weight markers; lane 12, starting extract; lane 13, DEAE I; lane 14, G-25 desalting column; lane 15, DEAE II; lane 16, G-25 desalting column; lane 17, DEAE III; lane 18, HIC; lane 19, DEAE IV. Six micrograms of protein were added to each gel lane. The gels were stained with Imperial protein stain (Pierce).

Fig. 3.

PON1 levels and protection after injection of rHuPON1_K192 into PON1^−/− mice. (A) Plasma PON1 DZOase levels (●) after i.p. injection of 1.12 units of rHuPON1_K192 into PON1^−/− mice (average of three mice). Maximal PON1 levels were approximately half those observed in wild-type mice, represented by the dashed line. Note the lack of DZOase in plasma of the saline-injected PON1^−/− mice (○). (B) Plasma PON1 DZOase levels after i.p. and i.m. injection of a total of 192 μg of rHuPON1_K192 (3.91 units) into each of two PON1^−/− mice. The dashed line represents DZOase activity in plasma of wild-type mice. Female and male mice injected with saline had no DZOase activity (○). DZOase of female (♦) and male (●) mice injected with rHuPON1 are shown separately. At 48 h after rHuPON1 injection, the mice were challenged with 1 mg/kg of DZO. (C) Injection of rHuPON1_K192 protected brain ChE from inhibition, measured 6 h after DZO exposure. Brain ChE activity was expressed as a percentage of untreated PON1^−/− mice (black bar). Inhibition in the rHuPON1_K192-injected mice is represented by the gray bar; inhibition in the saline-injected mice, by the white bar (average of two mice for each group).

Fig. 4.

Plasma PON1 levels over time after i.p./i.m. injection of 192 μg (3.91 units) of rHuPON1_K192 into each of two male PON1^−/− mice that had received DZO exposures of either 3 or 7 mg/kg 10 min before the rHuPON1 injection. None of the control mice displayed any plasma DZOase activity (○). One male mouse was challenged with 3 mg/kg of DZO, then injected with rHuPON1_K192 (♦); the other was treated with 7 mg/kg, then injected with the protein (●). The mice were euthanized at 6 h after DZO exposure for determination of brain ChE activity.